The present invention is related to the detection and/or treatment of cancer. More specifically, the present invention utilizes the existence of AFP receptor as a basis to detect cancer or contain or eliminate cancer in a patient.
Twenty years ago, Abelev et al. reported the existence of the first oncofetal antigen, alphafetoprotein (AFP) [Abelev, G. I., Perova, S. D., Khramkova, N. I., Postnikova, Z. A. and Irlin, I. S., Transplantation 1, 174 (1963)]. Although this is the major circulating protein during fetal life, it almost disappears after birth, its normal adult serum concentration being less than 50 ng/ml [Ruoslahti, E. and Seppals, M., Int. J. Cancer 8, 374 (1971)]. However, in certain malignant diseases such as hepatocarcinomas or teratocarcinomas, plasma levels can be one thousand-fold higher [Ruoslahti, E. and Seppals, M. Adv. Cancer Res. 29, 275 (1979)]. This finding not only drew the attention of clinicians, who envisaged a new means for detecting malignancy and monitoring cancer patients, but also the interest of investigators studying the physiology of that protein during fetal life.
One of the first parameters studied was AFP distribution within the embryo. Using immunoperoxidase methods, Benno and Williams described the distribution of AFP in the developing rat brain [Benno, R. H. and Williams, T. H., Brain Res. 142, 1982 (1978)]. Shortly thereafter, a series of papers reported the localization of plasma proteins within developing nervous cells in several species including birds and man [Trojan, J. and Uriel, J., J. Oncodevelop. Biol. Med. 1, 107 (1980); Uriel, J., Trojan, J., Dubouch, P. and Pieiro, A., Path. Biol. 30, 79 (1982); Moro, R. and Uriel, J., J. Oncodevelop. Biol. Med. 2, 391 (1981); Dziegielewska, K. M., Evans, C. A. N., Lorscheider, E. L., Malinowska, D. H., Mollgard, K., Reynolds, M. L. and Saunders, N. R., J. Physiol. 318, 239 (1981); Mollgard, K., Jacobsen, M., Krag-Jacobsen, G., Praetorius-Claussen, P. and Saunders, N. R., Neurosci. Lett. 14, 85 (1979)]. For a given tissue or organ, the kinetics of the staining for AFP and serum albumin (SA) follow a rather constant pattern across different species [Uriel, J., Trojan, J., Moro, R. and Pieiro, A., Ann. N.Y. Acad. Sci. 417, 321 (1983)]. When a nervous structure is very immature, no intracellular AFP or SA is detected. Then, suddenly, and for a certain period of time depending upon the species, both proteins are simultaneously observed, even within the same cell [Torand-Allerand, C. D., Nature 286, 733 (1980)]. Subsequently, staining intensity fades and positive cells become scarce, first for AFP and later for SA. Mature structures are negative for both proteins. Other serum constituents, such as IgG, or ovalbumin in chicken embryos, are never found during fetal life within neural cells, in spite of being present in the cerebrospinal fluid [Fielitz, W., Esteves, A. and Moro, R., Dev. Brain Res. 13, 111 (1984)].
Incorporation of AFP by Embryonic Cells
One question arising from these initial observations was whether AFP and SA were incorporated from extracellular sources or synthesized in-situ. While it is not yet clear if neural cells are capable of synthesizing plasma proteins [Ali, M., Raul, H. and Sahib, M., Dev. Brain Res. 1, 618 (1981); Ali, M., Mujoo, K. and Sahib, M., Dev. Brain Res. 6, 47 (1983); Schachter, B. S. and Toran-Allerand, C. D., Dev. Brain Res. 5, 95 (1982); Pieiro, A., Calvo, M., Iguaz, F., Lampreave, F. and Naval, J. Int. J. Biochem. 14, 817 (1982)], it has been demonstrated, both in-vitro [Uriel, J., Faivre-Bauman, A., Trojan, J. and Foiret, D. Neurosci. Lett. 27, 171 (1981); Hajeri-Germond, M., Trojan, Uriel, J. and Hauw, J. J. Dev. Neurosci. 6, 111 (1984)] and in-vivo [Villacampa, M. J., Lampreave, F., Calvo, M., Pieiro, A. and Uriel, J. Dev. Brain Res. 12, 77 (1984); Moro, R., Fielitz, W., Grunberg, J. and Uriel, J., Int. J. Dev. Neurosci. 2, 143 (1984)], that neuroblasts can readily incorporate AFP and serum albumin from extracellular sources. The in-vivo experiments were done with homologous and with heterologous proteins. In the first case [Villacampa, M. J., Lampreave, F., Calvo, M., Pieiro, A. and Uriel, J. Dev. Brain Res. 12, 77 (1984)], it was shown that upon injection into pregnant rats, 125I-AFP localized in the fetal brain, as well as in other fetal organs such as the gut, skin, and tongue, organs in which native intracellular AFP had been previously reported [Trojan, J. and Uriel, J., Oncodev. Biol. Med. 3, 13 (1982)]. The second set of experiments [Moro, R., Fielitz, W., Grunberg, J. and Uriel, J., Int. J. Dev. Neurosci. 2, 143 (1984)] showed that when newborn rat serum was injected into the mesencephalic cavity of chicken embryos, rat AFP and rat SA could be detected in the same location as their native counterparts. This also indicated that AFP and SA from one species are taken up by cells from another species, thus pointing to structures and mechanisms conserved throughout evolution. This, in turn, suggests a basic biological principle is involved.
In spite of the high concentration of rat IgG injected, the staining for this protein was negative. This is not the result of its high molecular weight (150,000) which could hinder a passive diffusion, since ovalbumin (MW=43,000) could not be detected either, even when injected at twofold the normal molar concentration of AFP in the embryonic cerebrospinal fluid [Fielitz, W., Esteves, A. and Moro, R., Dev. Brain Res. 13, 111 (1984)]. This selectivity favoured the hypothesis of a specific receptor mediated mechanism of endocytosis [Moro, R. and Uriel, J., J. Oncodevelop. Biol. Med. 2, 391 (1981); Moro, R., Fielitz, W., Grunberg, J. and Uriel, J., Int. J. Dev. Neurosci. 2, 143 (1984)].
AFP Incorporation Depends on Cell Differentiation
However, at this point it was still unclear whether the uptake of AFP and SA was a cell-dependent phenomenon, or if the staining disappeared as a result of low extracellular protein availability due to the closing of the brain-blood barrier or to the low concentration of circulating AFP at the end of fetal life. It was demonstrated, first in chicken [Moro, R., Neurosci. Lett. 41, 253 (1983)] and then in human embryos [Jacobsen, M., Lassen, L. C. and Mollgard, K., Tumor Biol. 5, 55 (1984)], that spinal ganglion neural cells accomplish the entire negative-positive-negative staining cycle for AFP before its highest peak in serum is attained. Moreover, when AFP becomes undetectable, SA continues to be present for some time, thus indicating that these serum proteins have access to the ganglionic neuroblasts.
AFP Receptors in Immature Cells
The cellular uptake of AFP suggests the existence of a specific receptor whose expression is regulated according to the degree of cell differentiation [[Uriel, J., Trojan, J., Moro, R. and Pieiro, A., Ann. N.Y. Acad. Sci. 417, 321 (1983); Moro, R., Neurosci. Lett. 41, 253 (1983)]. A previous report [Uriel, J., Bouillon, D., Russel, C. and Dupiers, M., Proc. Nat. Acad. Sci. U.S.A. 73, 1452 (1976)] showed the presence of two ultracentrifugation fractions containing AFP in immature rat uterine cytosols; a 4s fraction, corresponding entirely to AFP, and an 8s fraction in which the immunological detection of AFP was only possible after treatment with 0.4 M KCI. This treatment transformed the 8s fraction into the 4s one. Very likely the 8s fraction corresponded to a receptor-AFP complex, which was dissociated at high KC1 concentrations, even though at the time the AFP receptor concept was not born yet. This dissociation of the AFP-receptor complex with KC1 was also observed by Smalley and Sarcione [Smalley, J. R. and Sarcione, E. J. Bioch. Biohys. Res. Comm. 94, 1429 (1980)] who also provided evidence that the AFP molecule could be synthesized by immature rat uterus cells.
Expression of the AFP Receptor in Cancer Cells
Since cancer cells share a number of common biochemical and antigenical features with fetal cells [Uriel, J., Adv. Cancer Res. 29, 127 (1979)], it is possible that malignant cells, derived from tissues which incorporate AFP during fetal life, might reexpress the corresponding receptor and thus take up AFP again. In support of this hypothesis, Sarcione et al., [Sarcione, E. J., Zloty, M., Delluomo, D. S., Mizejewski, G. and Jacobson, H., Cancer Res. 43, 3739 (1983)] found AFP in an 8s complex derived from human mammary carcinomas which could be dissociated by KC1 treatment in the same way as in the experiments using immature rat cytosols. More recently, these authors have demonstrated that AFP is synthesized by the MCF-7 human breast cancer cell line as a complex which needs to be dissociated in order to make AFP immunologically detectable [Sarcione, E. J. and Hart, D., Int. J. Cancer 35, 315 (1985)]. On the other hand, this cell line [Uriel, J., Failly-Crepin, C., Villacampa, M. J., Pieiro, A., and Geuskens, M., Tumor Biol. 5, 41 (1984)], and a nickel induced rat rhabdomyosarcoma [Uriel, J., Poupon, M. F. and Geuskens, M., Br. J. Cancer 48, 263 (1983)] were shown to take up AFP in vitro. As a confirmation of these indirect results, surface receptors for AFP were detected on the MCF-7 line [Villacampa, M. J., Moro, R., Naval, J., Failly-Crpin, Ch., Lampreave, F. and Uriel, J., Bioch. Biophys. Res. Commun. 122, 1322 (1984)]. The binding parameters point to a two-site receptor model exhibiting positive cooperation. The high affinity site has a Kd of 1.5xc3x9710xe2x88x929 M with an exhibiting positive cooperation. The high affinity site has a Kd of 1.5xc3x9710xe2x88x929 M with an n-2,000/cell. The low affinity site, present at 320,000/cell has a Kd of 2.2xc3x9710xe2x88x927 M. Later studies showed the presence of a similar receptor system on the surface of mouse YACT lymphoma cells, which is absent from normal adult mouse T cells [Naval, J., Villacampa, M. J., Goguel, A. F. and Uriel, J. Proc. Natl. Acad. Sci. U.S.A. 82, 3301 (1985)].
These studies were done in parallel with in-vivo experiments in which mice bearing spontaneous mammary tumors were injected with radioiodinated AFP. The tissue distribution of radioactivity showed a tumor/normal tissue (liver) ratio of 3.6 [Uriel, J., Villacampa, M. J., Moro, R., Naval, J. and Failly-Crpin, CH. C.R. Acad. Sci. (Paris) 297, 589 (1983); Uriel, J., Villacampa, M. J., Moro, R., Naval, J. and Failly-Crpin, C., Cancer Res. 44, 5314 (1984)]. Autoradiography of tumor sections from these animals showed a significant accumulation of silver grains around the nuclear membrane of malignant cells but not of normal cells [Uriel, J., Villacampa, M. J., Moro, R., Naval, J. and Failly-Crpin, C., Cancer Res. 44, 5314 (1984)].
Scintigraphic Imaging of Mouse Tumors Using 131I-AFP
Using 131I-AFP, positive scintigraphic images of mouse mammary tumors as small as 3-4 mm have been obtained [Uriel, J., Villacampa, M. J., Moro, R., Naval, J. and Failly-Crpin, C., Cancer Res. 44, 5314 (1984); Moro, R., Heuguerot, C., Vercelli-Retta, J., Fielitz, W., Lpez, J. J. and Roca, R., Nuclear Med. Comm. 5, 5 (1984)]. In fact, eleven out of twelve such tumors were detectable with a standard gamma camera linked to a computer. Another mouse tumor, a neuroblastoma, could also be scanned in a similar manner [Hajeri-Germond, M., Naval, J., Trojan, J. and Uriel, J., Br. J. Cancer 51, 791 (1985)].
The expression of AFP uptake or direct evidence for the AFP receptor has been shown in several different types of tumors some of which are: A rat rhabdomyosarcoma [Uriel, J., Poupon, M. F. and Geuskens, M., Br. J. Cancer 48, 263 (1983)], a mouse neuroblastoma [Hajeri-Germond, M., Naval, J., Trojan, J. and Uriel, J., Br. J. Cancer 51, 791 (1985)], mouse and human mammary carcinomas [Villacampa, M. J., Moro, R., Naval, J., Failly-Crpin, Ch., Lampreave, F. and Uriel, J., Bioch. Biophys. Res. Commun. 122, 1322 (1984); Naval, J., Villacampa, M. J., Goguel, A. F. and Uriel, J. Proc. Natl. Acad. Sci. U.S.A. 82, 3301 (1985); Uriel, J., Villacampa, M. J., Moro, R., Naval, J. and Failly-Crpin, CH. C.R. Acad. Sci. (Paris) 297, 589 (1983); Uriel, J., Villacampa, M. J., Moro, R., Naval, J. and Failly-Crpin, C., Cancer Res. 44, 5314 (1984); Moro, R., Heuguerot, C., Vercelli-Retta, J., Fielitz, W., Lpez, J. J. and Roca, R., Nuclear Med. Comm. 5, 5 (1984); Biddle, W. and Sarcione, E. J., Breast Cancer Res. Treat. 10, 281 (1987)] mouse T lymphomas [Naval, J., Villacampa, M. J., Goguel, A. F. and Uriel, J. Proc. Natl. Acad. Sci. U.S.A. 82, 3301 (1985), human T and B cell lymphomas [Laborda, J., Naval, J., Allouche, M., Calvo, M., Georgoulias, V., Mishal, Z. and Uriel, J. Int J. Cancer 40, 314 (1987); Calvo, M., Laborda, J., Naval, J., Georgoulias, V. and Uriel, J., presented at the XIII Meeting of the ISOBM (Paris 1985); Torres, J. M., Anel, A., and Uriel, J., J. Cell Physiol. 150, 458 (1992); Torres, J. M., Gueskens, M. and Uriel, J., Int. J. Cancer 47, 112 (1991)] as well as phittohemaglutinin activated human T lymphocytes [Torres, J. M., Laborda, J., Naval, J., Darracq, N., Calvo, M., Mishal, Z. and Uriel, J. Mol. Immunology 26, 851 (1989)], the human malignant monocyte cell line U937 [Suzuki, Y., Zeng, C. Q., Alpert, E. J. Clinic. Invest. 90, 1530 (1992)] and the HT29 human colon carcinoma cell line [Esteban, C., Gueskens, M. and Uriel, J., Int. J. Cancer 49, 425 (1991)].
These findings place the AFP receptor as a widespread oncofetal antigen, related to the malignant state rather than the tumor type.
Monoclonal Antibodies Against the AFP Receptor
Labeled AFP (FITC, radioactive tracers) does not bind to tumor cells on paraffin tissue sections, probably due to the partial denaturation of the receptor""s binding site during fixation and to the relatively low affinity it exhibits towards its receptor. Thus, monoclonal antibodies (Mabs) against the AFP receptor were produced using a pool of human mammary carcinomas as the immunogen [Moro, R., Tamaoki, T., Wegmann, T. G., Longenecker, B. M., and Laderoute, M. P. Tumour Biol. 14, 116 (1993), incorporated by reference].
Two IgM producing Mabs recognize a 67 KD double band on PAGE gels under non-reducing conditions. The 67 KD bands are also reactive with 125I-AFP. These Mabs react with the binding site of the AFP receptor, since they inhibit the binding of AFP to tumor cells, and conversely they are inhibited from binding to the cells in the presence of a large excess of AFP. The Mabs do not react with AFP. They recognize fetal cells and mammary carcinomas on tissue sections, but not mammary adenomas or most other normal adult tissues.
During the last decades, scientists have been trying to characterize antigens related to malignancy. The AFP receptor, which should be considered as an oncofetal antigen could fulfill many of the requirements of a clinically useful tumor marker. Further work using monoclonal antibodies against this widespread cancer related antigen will allow one to determine their clinical usefulness as well as to study the physiological role of the AFP receptor.
The present invention pertains to a method for detecting cancer in a patient. The method comprises the steps of introducing labeled antibodies or labeled AFP to a biological sample of the patient so the labeled antibodies or labeled AFP will react with the AFP receptor binding sites in the biological sample. Next there is the step of identifying AFP receptor binding sites in the biological material which are reacted with the labeled antibodies or labeled AFP to determine the presence of cancer. Preferably, before the introducing step, there is the step of obtaining a biological sample from a body of a patient.
The present invention pertains to a method for treating cancer cells in a patient. The method comprises the steps of introducing AFP receptor antibodies to cancer cells in the patient. Then there is the step of reacting the AFP receptor antibodies with the AFP receptor of the cancer cells to inhibit growth of the cancer cells or kill the cancer cells.
The present invention pertains to a method for monitoring a patient. The method comprises the steps of treating the patient for cancer. Then there is the step of testing the patient at predetermined intervals after the treatment for AFP receptor site levels.
The present invention pertains to a method for treating a patient. The method comprises the steps of testing the patient for AFP receptor. Then there is the step of introducing AFP receptor antibodies or AFP into the patient to react with cancer cells in the patient if the testing indicates AFP receptors are present in the patient.
The present invention pertains to a method for treating cancer cells in a patient. The method comprises the steps of introducing modified AFP to cancer cells in the patient. Then there is the step of reacting the modified AFP with the AFP receptor of the cancer cells to inhibit growth of the cancer cells or kill the cancer cells.
It is the object of the invention to diagnose and follow up cancer diseases and pregnancy by detecting the alpha-fetoprotein receptor (AFP receptor) in bodily fluids and tissues. Even though the principles and methods are similar for detecting the AFP receptor in solution (bodily fluids) or attached to a solid matrix (tissue sections), they will be addressed separately for clarity purposes.